Maturation of the biochemical field has imposed demands on mass spectrometry (MS) for the analysis of increasingly large and complex biopolymers. Although spectrometer technology has profited from the electronics and computer revolution, two fundamental limitations remain. Analytes of interest must be presented as gas phase ions and their mass to charge ratio (m/z) must be within the instrumental mass range. While increasing mass range is relatively straight forward, producing gas phase ions of polar, nonvolatile and thermally fragile molecules is not.
With the exception of the now nearly abandoned method of Field Desorption (FD), most biomolecule ionization schemes are based on the application of tremendous energy in a short time period. These "energy sudden" methods such as Fast Atom Bombardment (FAB), Laser desorption (LD) and Plasma Desorption (PD), rely on power density to affect sample volatilization/ionization and analysis prior to analyte decomposition. While effective, these methods predominately produce singly charged ions, the analysis of which remains limited by the mass range of common multipurpose sector and quadrupole spectrometers. Further, the transient nature of the ion signal and/or the sample matrix requirements limit or preclude their application to on-line Liquid Chromatography Mass Spectrometry LC/MS.
Electrospray Ionization (ESI) is the latest development in LC/MS interfacing and one which addresses both the ionization and mass range problems. Like Thermospray (TSP), it is a soft spray ionization technique predicated on field induced "ion evaporation" from the surface of charged liquid droplets. Both techniques nebulize the analyte containing semi-aqueous mobile phase to form an aerosol of submicron droplets from which ion evaporation occurs. ESI differs from TSP in three respects;
1) The aerosol is generated electrostatically rather than thermo-pneumatically.
2) Nebulization/ionization is performed at atmospheric rather than reduced pressure.
3) Electrostatic nebulization limits flow rates to ca. 1 to 50 mcl/min.
These differences give ESI its inherently high ionization efficiency and hence mass sensitivity, its ability to produce large gas phase ions of very high charge state (m/z within instrumental mass range), and dictate the low flow regime in which the technique operates.
The current monumental interest in ESI is ironic considering it is the chronological predecessor of most of the energy sudden ionization "breakthroughs" developed over the past twenty years. While electrostatic aerosol generation is not new and has had numerous industrial applications, it was not applied to mass spectrometry until 1968 by M. Dole and others. M. Dole's interest in "macro-ions" of synthetic polymers and adherence to the now rejected "ion residue" model lead to his expectation of large singly charged ions beyond the mass range of analyzers of the day. For this reason, he examined energy analysis for mass determination. Unfortunately, due to the focus of his research, his choice of mass analyzer, and the comparative infancy of biochemistry, the implications of his "discovery" were not recognized by the MS community.
In the late seventies, M. L. Vestal and coworkers discovered TSP while investigating direct thermal LC/MS interfacing methods in ignorance of M. Dole's work. TSP produces charged droplets based solely on statistical charge segregation of buffer/analyte ions manifesting horrendously poor ionization efficiency. Although TSP sensitivity was viewed by many as inadequate for useful biopolymer applications, it did demonstrate the ability to produce ions of these species and with higher charge states. The current ion evaporation model was subsequently proposed.
The success of TSP and the need for higher ionization efficiency (sensitivity) led J. B. Fenn, C. M. Whitehouse and others in the mid eighties to reexamine M. Dole's electrostatic nebulization. Their focus on biopolymers and the availability of compatible high gas load quadrupole spectrometers met with immediate success and ESI became the latest MS bioanalytical bombshell. Commercial ESI interface designs were soon available. The interest in ESI is now immense as evidenced by the 1991 meeting of the American Society of Mass Spectrometry (ASMS) in which there were more than 100 presentations on ESI alone.
All ESI interface designs perform two basic functions; the electrostatic generation of an aerosol at atmospheric pressure from which gas phase analyte ions are produced by ion evaporation and the conveyance of these ions to the high vacuum of the spectrometer. Most designs only differ significantly in how the latter is accomplished.
Electrostatic aerosol generation differentiates ESI from TSP and accounts for most of its unique performance. Depending on the chromatographic scale, all or a fraction (1 to 50 mcl/min) of the semi-aqueous LC eluent is passed through a narrow stainless steel capillary "needle" held at 2 to 4 kV relative to a reference plate. Typically 28 to 33 gauge hypodermic needle stock is used for the capillary with the reference plate being the interface housing itself. Reverse configurations placing high voltage on the reference plate while grounding the needle have also been constructed to minimize electrical complications with chromatographic hardware. It is only important that the relative sense of the needle potential be the same as the ions to be generated.
The intense electrostatic field gradient established between the needle and reference plate is of maximum intensity at the needle tip, approximated by a point electrode. The net electrostatic forces acting on solution state ions elongates the emanating droplet to form a "Taylor" cone. For example, with positive ion MS, solution state cations are attracted toward the relatively negative ground plate while anionic species are electrochemically neutralized at the needle surface leaving predominately cations in the Taylor cone. In this respect, ESI provides charge pumping.
Electrostatic force draws the cone apex into a charged fluid filament a few microns in diameter. Electrostatic repulsion causes ion migration to the filament surface. As the applied field strength decreases with distance from the needle tip, ionic repulsion overcomes surface tension, dispersing the filament into an aerosol of submicron highly charged droplets. The nature of this mechanism dictates that droplets are formed at or near the Raleigh limit--the point at which surface charge repulsion is balanced by surface tension.
Droplets so formed are desolvated as they traverse the atmospheric pressure region between the needle and plate with the heat of vaporization efficiently provided by the ambient gas. Desolvation reduces droplet radii creating surface field strengths above the Raleigh limit. Droplets undergo a "coulombic explosion" to form secondary hyperfine charged droplets and to re-establish equilibrium. Many cycles of this desolvation/fragmentation process produce minute droplets of high net charge. These processes have recently been confirmed by high speed laser photography.
The near point charge concentration of the final droplets may be viewed as Field Desorption emitters. Surface field strengths well into the kilovolt range have been calculated. Solution analyte ions at the surface of these droplets are ejected directly into gas phase when electrostatic repulsion exceeds the solvation energy. This process is referred to as Ion Evaporation.
Although both ESI and TSP produce gas phase ions by ion evaporation, ESI is differentiated from its predecessor TSP by electrostatic aerosol generation at atmospheric pressure. TSP produces an aerosol by partial thermal vaporization of the mobile phase (flow rates 0.5 to 3.0 ml/min) in a metal capillary. Droplets are produced by the pneumatic expansion of the liquid/vapor exiting the capillary tip into a reduced pressure (10-50 torr) source region. Net charging of TSP droplets relies on the random statistical segregation of solution buffer and analyte ions in individual droplets. Both cationic and anionic droplets are produced with ensemble neutrality. Electrostatic charging is not viable at low pressure due to the early onset of corona.
TSP nebulization produces droplets of larger initial radius and much lower net charge as predicted by common sense and proven by Faraday current measurements. Initial droplets are produced well below the Raleigh limit requiring significant desolvation to induce coulombic fragmentation and ion evaporation. Adiabatic expansion and evaporative cooling in the reduced pressure source arrests droplet desolvation requiring copious heating of the TSP source block. Poor thermal contact at low pressure limits desolvation. Low initial charging and incomplete droplet desolvation make the ionization efficiency and hence sensitivity of TSP markedly lower than ESI.
Atmospheric aerosol generation in ESI has ramifications beyond providing the heat of vaporization. The translational, rotational and internal energies of the resulting gas phase ions are defined by the temperature of the ambient bath gas. In this region, energetically "cold" evaporated ions do not participate in ion/molecule processes in the conventional sense. Although Atmospheric Pressure Chemical Ionization (APCI) is well known and useful, it is initiated by very energetic corona or plasma primary ionization with analyte ionization by energy transfer occurring within microseconds during thermalization. Evaporated ions do not possess this initial energy. The influence on this mechanism is not well understood for ESI, but remains important. The higher charge state of ESI ions likely results from the initial charge pumping of the aerosol droplets as well as the inability of these ions to lose charge to thermalized collision partners at atmospheric pressure.
ESI also produces little matrix background from mobile phase constituents. The intense solvent/buffer ion clusters associated with TSP are not observed because their formation mechanism may be restricted by ion evaporation at high pressure. The reduction of chemical interference from the mobile phase is equally as important as the inherent ionization efficiency of ESI. Combined, the two yield a signal-to-noise enhancement of greater than 1000 over TSP on a mass flux basis. This performance is mandatory for successful interfacing to conventional (ml/min) HPLC systems because most of these gains are offset by the requisite splitting of the column eluent. TSP consumes the entire HPLC eluent.
The ESI interface performs three basic functions; 1) It defines the aerosol generation chamber and maintains the electrostatic and thermal conditions needed for efficient and stable ion production. 2) It provides for "declustering" of adducted solvent molecules from gas phase analyte ions. 3) It conveys analyte ions from atmospheric pressure to the high vacuum of the mass analyzer. To accomplish these, two basic interface designs have emerged; the coaxial conical skimmer and the coaxial capillary drift tube. Both designs rely on momentum separation to enrich heavier analyte ions from lighter atmospheric bath gas constituents.
The conical skimmer scheme has existed for decades having been initially developed for high energy corona and plasma APCI applications. A series of two or more conical skimmer cones are arranged behind a forward aperture and coaxial with the gas flow to form the momentum separator. A cylindrical tube about the forward plate defines the aerosol volume. The aerosol volume and the separator are heated independently.
With ESI, the forward aperture plate acts as the planar counter electrode to the aerosol needle. Dry nitrogen curtain gas is heated as it passes between the forward plate and the first skimmer and emerges countercurrent to the aerosol flow. This "drying gas" promotes desolvation of the aerosol droplets and declustering of adducted solvent molecules from evaporated analyte ions. Analyte ions and ambient bath gas are drawn into the first skimmer by instrument vacuum with the pressure between the skimmers reduced by mechanical rotary pumps. Heavier analyte ions maintain their trajectory through the skimmers to enter the mass analyzer while lighter mobile phase and bath gas species are collisionally scattered and pumped away. Voltage placed on the skimmer cones may be used to achieve collisional activation in this reduced pressure region. In addition to diffusion or turbomolecular pumping, cryo-pumping is employed to achieve a final analyzer vacuum of ca. 10.sup.-6 torr.
Considerable research went into the optimization of skimmer cone geometry and placement with respect to the expansion mach cone in order to maximize ion transmission. A highly successful design of J. A. Buckley et al (U.S. Pat. No. 4 148 196), developed at the University of Toronto, is marketed by SCIEX Ltd. of Canada as an integral component of their quadrupole APCI systems. These systems offer excellent ESI performance. The disadvantages of the design are that it is a relatively complex and dedicated system with inordinately large pumping requirements.
The capillary drift tube design is functionally identical with the aerosol volume and forward aperture plate being similarly constructed. Analyte ions and bath gas are drawn into the interface by instrument vacuum via a heated metal or metalized fused silica capillary drift tube located concentrically within the forward aperture. Countercurrent drying gas is introduced between the capillary and forward aperture for the same purpose as before. Ions traversing the 20 cm by 0.5 mm ID capillary are thermally declustered before entering the single stage mechanically pumped momentum separator. Heavier analyte ions are again conveyed to the analyzer via a single skimmer cone while lighter solvent and bath gas species are pumped away. Bias voltage placed on the capillary accelerates ions exiting the drift tube to provide collisional activation within the skimmer region. Gas flow into the interface is limited by the conductance of the drift tube such that conventional diffusion or turbomolecular analyzer pumping is adequate.
The design of Fenn, Meng and Mann (WO90/14148) has been commercially implemented on Finnigan quadrupole spectrometer systems. Chowdhury, Katta and Chait have incorporated a similar design (U.S. Pat. No. 4 977 320) on Hewlett-Packard MSD bench top systems which does not make use of the drying gas. Both devices have demonstrated admirable ESI performance. These designs have the advantage of simplicity and reduced pumping requirements, however, they remain integral components requiring significant modification and hence near dedication of the spectrometer.
The uniqueness of ESI as a primary ionization method and as an LC/MS interface make its application to pharmaceutical development problems highly desirable. Due to the high cost and instrumental dedication required by commercially available devises, the development of a simple, cost effective, yet functional interface more amenable to the multi-mode research MS environment was undertaken. The provision of a simple probe based ESI interface system fulfilling these requirements is the subject matter of this invention.
Accordingly, it is an object of this invention to provide a device for facilitating the conversion of a thermospray equipped mass spectrometry analyzer to one capable of using electrospray technology without necessitating a removal of the thermospray equipment.
It is a further object of this invention to provide a probe-based electrospray adapter for use in converting a thermospray equipped quadrupole based LC/MS analyzer to an analyzer capable of receiving ions created by electrospray ionization technology.
It is a further object of this invention to provide a probe, as aforesaid, capable of use with an existing thermospray equipped LC/MS analyzer without complicated changes being required to the analyzer to thereby facilitate a continued and convenient use of all instrument capabilities at a subsequent time.
It is a further object of this invention to provide a probe, as aforesaid, which has adjustable features on it that can enhance the responsiveness of the analyzer, which adjustment features are independent of structure of the analyzer.
It is a further object of this invention to provide a probe, as aforesaid, which is inexpensive to manufacture, easy to operate and easy to maintain in proper operative condition to achieve high quality results from the analyzer.